Method of identifying a compound for inhibiting or stimulating human G protein-coupled receptors

ABSTRACT

The invention disclosed in this patent document relates to transmembrane receptors, more particularly to endogenous, human orphan G protein-coupled receptors.

This application is a continuation of Application Ser. No. 10/272,983,filed Oct. 17, 2002, which is a continuation of Application Ser. No.09/417,044, filed Oct. 12, 1999, now abandoned, and claims prioritybenefit of Provisional Application Ser. No. 60/121,852 filed Feb. 26,1999, Ser. Provisional Application No. 60/109,213, filed Nov. 20, 1998,Ser. Provisional Application No. 60/120,416, filed Feb. 16, 1999, Ser.Provisonal Application No. 60/123,946, filed Mar. 12, 1999, Ser.Provisional Application No. 60/123,949, filed Mar. 12, 1999, Ser.Provisonal Application No. 60/136,436, filed May 28, 1999, Ser.Provisional Application No. 60/136,439, field filed May 28, 1999, Ser.Provisional Application No. 60/136,567, file filed May 28, 1999, Ser.Provisional Application No. 60/137,127, filed May 28, 1999, Ser.Provisional Application No. 60/137,131, filed May 28, 1999, Ser.Provisional Application No. 60/141,448, filed Jun. 29, 1999, Ser.Provisional Application No. 60/136,437, filed May 28, 1999, Ser.Provisional Application No. 60/156,653, filed Sep. 29, 1999, Ser.Provisional Application No. 60/156,333 60/156,633, filed Sep. 28 29,1999, Ser. Provisional Application No. 60/156,555, filed Sep. 29, 1999,Ser. Provisional Application No. 60/156,634, filed Sep. 29, 1999, Ser.Provisional Application No. 60/157,280, filed Oct. 1, 1999, Ser.Provisional Application No. 60/157,294, filed Oct. 1, 1999, Ser.Provisional Application No. 60/157,281, filed Oct. 1, 1999, Ser.Provisional Application No. 60/157,293, filed Oct. 1, 1999, and Ser.Provisional Application No. 60/157,282, filed Oct. 1, 1999, the entiretyof each of which is incorporated herein by reference. This patentapplication is related to U.S. Ser. Application No. 09/170,496, filedOct. 13, 1999, and U.S. Ser. Application No. 09/416,760, filed Oct. 12,1999, both being incorporated herein by reference in their entirety.This patent application is also related to U.S. Ser. Application No.09/364,425, filed Jul. 30, 1999, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The invention disclosed in this patent document relates to transmembranereceptors, and more particularly to endogenous, orphan, human Gprotein-coupled receptors (“GPCRs”).

BACKGROUND OF THE INVENTION

Although a number of receptor classes exist in humans, by far the mostabundant and therapeutically relevant is represented by the Gprotein-coupled receptor (GPCR or GPCRs) class. It is estimated thatthere are some 100,000 genes within the human genome, and of these,approximately 2% or 2,000 genes, are estimated to code for GPCRs.Receptors, including GPCRs, for which the endogenous ligand has beenidentified are referred to as “known” receptors, while receptors forwhich the endogenous ligand has not been identified are referred to as“orphan” receptors. GPCRs represent an important area for thedevelopment of pharmaceutical products: from approximately 20 of the 100known GPCRs, 60% of all prescription pharmaceuticals have beendeveloped. This distinction is not merely semantic, particularly in thecase of GPCRs. Thus, the orphan GPCRs are to the pharmaceutical industrywhat gold was to California in the late 19^(th) century—an opportunityto drive growth, expansion, enhancement and development.

GPCRs share a common structural motif. All these receptors have sevensequences of between 22 to 24 hydrophobic amino acids that form sevenalpha helices, each of which spans the membrane (each span is identifiedby number, i.e., transmembrane-1 (TM-1), transmembrane-2 (TM-2), etc.).The transmembrane helices are joined by strands of amino acids betweentransmembrane-2 and transmembrane-3, transmembrane-4 andtransmembrane-5, and transmembrane-6 and transmembrane-7 on theexterior, or “extracellular” side, of the cell membrane (these arereferred to as “extracellular” regions 1, 2 and 3 (EC-1, EC-2 and EC-3),respectively). The transmembrane helices are also joined by strands ofamino acids between transmembrane-1 and transmembrane-2, transmembrane-3and transmembrane-4, and transmembrane-5 and transmembrane-6 on theinterior, or “intracellular” side, of the cell membrane (these arereferred to as “intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3),respectively). The “carboxy” (“C”) terminus of the receptor lies in theintracellular space within the cell, and the “amino” (“N”) terminus ofthe receptor lies in the extracellular space outside of the cell.

Generally, when an endogenous ligand binds with the receptor (oftenreferred to as “activation” of the receptor), there is a change in theconformation of the intracellular region that allows for couplingbetween the intracellular region and an intracellular “G-protein.” Ithas been reported that GPCRs are “promiscuous” with respect to Gproteins, i.e., that a GPCR can interact with more than one G protein.See, Kenakin, T, 43 Life Sciences 1095 (1988). Although other G proteinsexist, currently, Gq, Gs, Gi, and Go are G proteins that have beenidentified. Endogenous ligand-activated GPCR coupling with the G-proteinbegins a signaling cascade process (referred to as “signaltransduction”). Under normal conditions, signal transduction ultimatelyresults in cellular activation or cellular inhibition. It is thoughtthat the IC-3 loop as well as the carboxy terminus of the receptorinteract with the G protein.

Under physiological conditions, GPCRs exist in the cell membrane inequilibrium between two different conformations: an “inactive” state andan “active” state. A receptor in an inactive state is unable to link tothe intracellular signaling transduction pathway to produce a biologicalresponse. Changing the receptor conformation to the active state allowslinkage to the transduction pathway (via the G-protein) and produces abiological response. A receptor may be stabilized in an active state byan endogenous ligand or a compound such as a drug.

SUMMARY OF THE INVENTION

Disclosed herein are human endogenous orphan G protein-coupledreceptors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B provide reference “grids” for certain dot-blots providedherein (see also, FIGS. 2A and 2B, respectively).

FIGS. 2A and 2B provide reproductions of the results of certain dot-blotanalyses resulting from hCHN3 and hCHN8, respectively (see also, FIGS.1A and 1B, respectively).

FIG. 3 provides a reproduction of the results of RT-PCR analysis ofhRUP3.

FIG. 4 provides a reproduction of the results of RT-PCR analysis ofhRUP4.

FIG. 5 provides a reproduction of the results of RT-PCR analysis ofhRUP6.

FIG. 6 is a reproduction of a photograph of the results of the tissuedistribution of RUP3 using multiple tissue (human) cDNA. Based uponthese tissues, the data support the position that RUP3 is expressed onlyin the pancreas.

DETAILED DESCRIPTION

The scientific literature that has evolved around receptors has adopteda number of terms to refer to ligands having various effects onreceptors. For clarity and consistency, the following definitions willbe used throughout this patent document. To the extent that thesedefinitions conflict with other definitions for these terms, thefollowing definitions shall control:

AMINO ACID ABBREVIATIONS used herein are set out in Table 1:

TABLE 1 ALANINE ALA A ARGININE ARG R ASPARAGINE ASN N ASPARTIC ACID ASPD CYSTEINE CYS C GLUTAMIC ACID GLU E GLUTAMINE GLN Q GLYCINE GLY GHISTIDINE HIS H ISOLEUCINE ILE I LEUCINE LEU L LYSINE LYS K METHIONINEMET M PHENYLALANINE PHE F PROLINE PRO P SERINE SER S THREONINE THR TTRYPTOPHAN TRY W TYROSINE TYR Y VALINE VAL V

COMPOSITION means a material comprising at least one component.

ENDOGENOUS shall mean a material that a mammal naturally produces.ENDOGENOUS in reference to, for example and not limitation, the term“receptor,” shall mean that which is naturally produced by a mammal (forexample, and not limitation, a human) or a virus. By contrast, the termNON-ENDOGENOUS in this context shall mean that which is not naturallyproduced by a mammal (for example, and not limitation, a human) or avirus.

HOST CELL shall mean a cell capable of having a Plasmid and/or Vectorincorporated therein. In the case of a prokaryotic Host Cell, a Plasmidis typically replicated as a autonomous molecule as the Host Cellreplicates (generally, the Plasmid is thereafter isolated forintroduction into a eukaryotic Host Cell); in the case of a eukaryoticHost Cell, a Plasmid is integrated into the cellular DNA of the HostCell such that when the eukaryotic Host Cell replicates, the Plasmidreplicates. Preferably, for the purposes of the invention disclosedherein, the Host Cell is eukaryotic, more preferably, mammalian, andmost preferably selected from the group consisting of 293, 293T andCOS-7 cells.

LIGAND shall mean an endogenous, naturally occurring molecule specificfor an endogenous, naturally occurring receptor.

MUTANT or MUTATION in reference to an endogenous receptor's nucleic acidand/or amino acid sequence shall mean a specified change or changes tosuch endogenous sequences such that a mutated form of an endogenous,non-constitutively activated receptor evidences constitutive activationof the receptor. In terms of equivalents to specific sequences, asubsequent mutated form of a human receptor is considered to beequivalent to a first mutation of the human receptor if (a) the level ofconstitutive activation of the subsequent mutated form of the receptoris substantially the same as that evidenced by the first mutation of thereceptor; and (b) the percent sequence (amino acid and/or nucleic acid)homology between the subsequent mutated form of the receptor and thefirst mutation of the receptor is at least about 80 %, more preferablyat least about 90 % and most preferably at least 95 %. Ideally, andowing to the fact that the most preferred mutation disclosed herein forachieving constitutive activation includes a single amino acid and/orcodon change between the endogenous and the non-endogenous forms of theGPCR, the percent sequence homology should be at least 98 %.

NON-ORPHAN RECEPTOR shall mean an endogenous naturally occurringmolecule specific for an endogenous naturally occurring ligand whereinthe binding of a ligand to a receptor activates an intracellularsignaling pathway.

ORPHAN RECEPTOR shall mean an endogenous receptor for which theendogenous ligand specific for that receptor has not been identified oris not known.

PLASMID shall mean the combination of a Vector and cDNA. Generally, aPlasmid is introduced into a Host Cell for the purposes of replicationand/or expression of the cDNA as a protein.

VECTOR sin reference to cDNA shall mean a circular DNA capable ofincorporating at least one cDNA and capable of incorporation into a HostCell.

The order of the following sections is set forth for presentationalefficiency and is not intended, nor should be construed, as a limitationon the disclosure or the claims to follow.

Identification of Human GPCRs

The efforts of the Human Genome project have led to the identificationof a plethora of information regarding nucleic acid sequences locatedwithin the human genome; it has been the case in this endeavor thatgenetic sequence information has been made available without anunderstanding or recognition as to whether or not any particular genomicsequence does or may contain open-reading frame information thattranslate human proteins. Several methods of identifying nucleic acidsequences within the human genome are within the purview of those havingordinary skill in the art. For example, and not limitation, a variety ofGPCRs, disclosed herein, were discovered by reviewing the GenBank™database, while other GPCRs were discovered by utilizing a nucleic acidsequence of a GPCR, previously sequenced, to conduct a BLAST™ search ofthe EST database. Table A, below, lists the disclosed endogenous orphanGPCRs along with a GPCR's respective homologous GPCR:

TABLE A Open Reference To Disclosed Reading Percent Homologous HumanAccession Frame Homology To GPCR Orphan Number (Base Designated(Accession GPCRs Identified Pairs) GPCR No.) hARE-3 AL033379 1,260 bp52.3% LPA-R U92642 hARE-4 AC006087 1,119 bp 36% P2Y5 AF000546 hARE-5AC006255 1,104 bp 32% Oryzias D43633 latipes hGPR27 AA775870 1,128 bphARE-1 AI090920   999 bp 43% D13626 KIAA0001 hARE-2 AA359504 1,122 bp53% GPR27 hPPR1 H67224 1,053 bp 39% EBI1 L31581 hG2A AA754702 1,113 bp31% GPR4 L36148 hRUP3 AI035423 1,005 bp 30% 2133653 Drosophilamelanogaster hRUP4 AI307658 1,296 bp 32% pNPGPR NP_004876 28% and 29%AAC41276 Zebra fish and Ya and Yb, AAB94616 respectively hRUP5 AC0058491,413 bp 25% DEZ Q99788 23% FMLPR P21462 hRUP6 AC005871 1,245 bp 48%GPR66 NP_006047 hRUP7 AC007922 1,173 bp 43% H3R AF140538 hCHN3 EST 365811,113 bp 53% GPR27 hCHN4 AA804531 1,077 bp 32% thrombin 4503637 hCHN6EST 2134670 1,503 bp 36% edg-1 NP_001391 hCHN8 EST 764455 1,029 bp 47%D13626 KIAA0001 hCHN9 EST 1541536 1,077 bp 41% LTB4R NM_000752 hCHN10EST 1365839 1,055 bp 35% P2Y NM_002563

Receptor homology is useful in terms of gaining an appreciation of arole of the disclosed receptors within the human body. Additionally,such homology can provide insight as to possible endogenous ligand(s)that may be natural activators for the disclosed orphan GPCRs.

B. Receptor Screening

Techniques have become more readily available over the past few yearsfor endogenous-ligand identification (this, primarily, for the purposeof providing a means of conducting receptor-binding assays that requirea receptor's endogenous ligand) because the traditional study ofreceptors has always proceeded from the a priori assumption(historically based) that the endogenous ligand must first be identifiedbefore discovery could proceed to find antagonists and other moleculesthat could affect the receptor. Even in cases where an antagonist mighthave been known first, the search immediately extended to looking forthe endogenous ligand. This mode of thinking has persisted in receptorresearch even after the discovery of constitutively activated receptors.What has not been heretofore recognized is that it is the active stateof the receptor that is most useful for discovering agonists, partialagonists, and inverse agonists of the receptor. For those diseases whichresult from an overly active receptor or an under-active receptor, whatis desired in a therapeutic drug is a compound which acts to diminishthe active state of a receptor or enhance the activity of the receptor,respectively, not necessarily a drug which is an antagonist to theendogenous ligand. This is because a compound that reduces or enhancesthe activity of the active receptor state need not bind at the same siteas the endogenous ligand. Thus, as taught by a method of this invention,any search for therapeutic compounds should start by screening compoundsagainst the ligand-independent active state.

As is known in the art, GPCRs can be “active” in their endogenous stateeven without the binding of the receptor's endogenous ligand thereto.Such naturally-active receptors can be screened for the directidentification (i.e., without the need for the receptor's endogenousligand) of, in particular, inverse agonists. Alternatively, the receptorcan be “activated” via, e.g., mutation of the receptor to establish anon-endogenous version of the receptor that is active in the absence ofthe receptor's endogenous ligand.

Screening candidate compounds against an endogenous or non-endogenous,constitutively activated version of the human orphan GPCRs disclosedherein can provide for the direct identification of candidate compoundswhich act at this cell surface receptor, without requiring use of thereceptor's endogenous ligand. By determining areas within the body wherethe endogenous version of human GPCRs disclosed herein is expressedand/or over-expressed, it is possible to determine relateddisease/disorder states which are associated with the expression and/orover-expression of the receptor; such an approach is disclosed in thispatent document.

With respect to creation of a mutation that may evidence constitutiveactivation of human orphan GPCRs disclosed herein is based upon thedistance from the proline residue at which is presumed to be locatedwithin TM6 of the GPCR typically nears the TM6/IC3 interface (suchproline residue appears to be quite conserved). By mutating the aminoacid residue located 16 amino acid residues from this residue(presumably located in the IC3 region of the receptor) to, mostpreferably, a lysine residue, such activation may be obtained. Otheramino acid residues may be useful in the mutation at this position toachieve this objective.

C. Disease/Disorder Identification and/or Selection

Preferably, the DNA sequence of the human orphan GPCR can be used tomake a probe for (a) dot-blot analysis against tissue-mRNA, and/or (b)RT-PCR identification of the expression of the receptor in tissuesamples. The presence of a receptor in a tissue source, or a diseasedtissue, or the presence of the receptor at elevated concentrations indiseased tissue compared to a normal tissue, can be preferably utilizedto identify a correlation with a treatment regimen, including but notlimited to, a disease associated with that disease. Receptors canequally well be localized to regions of organs by this technique. Basedon the known functions of the specific tissues to which the receptor islocalized, the putative functional role of the receptor can be deduced.

As the data below indicate, RUP3 is expressed within the human pancreas,suggesting that RUP3 may play a role in insulin regulation and/orglucagon regulation. Accordingly, candidate compounds identified using aconstitutively activated form of RUP3 may be useful for understandingthe role of RUP3 in diabetes and/or as therapeutics for diabetes.

D. Screening of Candidate Compounds

1. Generic GPCR Screening Assay Techniques

When a G protein receptor becomes constitutively active (i.e., active inthe absence of endogenous ligand binding thereto), it binds to a Gprotein (e.g., Gq, Gs, Gi, Go) and stimulates the binding of GTP to theG protein. The G protein then acts as a GTPase and slowly hydrolyzes theGTP to GDP, whereby the receptor, under normal conditions, becomesdeactivated. However, constitutively activated receptors continue toexchange GDP to GTP. A non-hydrolyzable analog of GTP, [³⁵S]GTPγS, canbe used to monitor enhanced binding to membranes which expressconstitutively activated receptors. It is reported that [³⁵S] GTPγS canbe used to monitor G protein coupling to membranes in the absence andpresence of ligand. An example of this monitoring, among other exampleswell-known and available to those in the art, was reported by Traynorand Nahorski in 1995. The preferred use of this assay system is forinitial screening of candidate compounds because the system isgenerically applicable to all G protein-coupled receptors regardless ofthe particular G protein that interacts with the intracellular domain ofthe receptor.

2. Specific GPCR Screening Assay Techniques

Once candidate compounds are identified using the “generic” Gprotein-coupled receptor assay (i.e., an assay to select compounds thatare agonists, partial agonists, or inverse agonists), further screeningto confirm that the compounds have interacted at the receptor site ispreferred. For example, a compound identified by the “generic” assay maynot bind to the receptor, but may instead merely “uncouple” the Gprotein from the intracellular domain.

a. Gs and Gi.

Gs stimulates the enzyme adenylyl cyclase. Gi (and Go), on the otherhand, inhibit this enzyme. Adenylyl cyclase catalyzes the conversion ofATP to cAMP; thus, constitutively activated GPCRs that couple the Gsprotein are associated with increased cellular levels of cAMP. On theother hand, constitutively activated GPCRs that couple the Gi (or Go)protein are associated with decreased cellular levels of cAMP. See,generally, “Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, FromNeuron To Brain (3^(rd)Ed.) Nichols, J. G. et al eds. SinauerAssociates, Inc. (1992). Thus, assays that detect cAMP can be utilizedto determine if a candidate compound is, e.g., an inverse agonist to thereceptor (i.e., such a compound would decrease the levels of cAMP). Avariety of approaches known in the art for measuring cAMP can beutilized; a most preferred approach relies upon the use of anti-cAMPantibodies in an ELISA-based format. Another type of assay that can beutilized is a whole cell second messenger reporter system assay.Promoters on genes drive the expression of the proteins that aparticular gene encodes. Cyclic AMP drives gene expression by promotingthe binding of a cAMP-responsive DNA binding protein or transcriptionfactor (CREB) which then binds to the promoter at specific sites calledcAMP response elements and drives the expression of the gene. Reportersystems can be constructed which have a promoter containing multiplecAMP response elements before the reporter gene, e.g., β-galactosidaseor luciferase. Thus, a constitutively activated Gs-linked receptorcauses the accumulation of cAMP that then activates the gene andexpression of the reporter protein. The reporter protein such asβ-galactosidase or luciferase can then be detected using standardbiochemical assays (Chen et al. 1995).

Go and Gq.

Gq and Go are associated with activation of the enzyme phospholipase C,which in turn hydrolyzes the phospholipid PIP₂, releasing twointracellular messengers: diacycloglycerol (DAG) and inistol1,4,5-triphoisphate (IP₃). Increased accumulation of IP₃ is associatedwith activation of Gq- and Go-associated receptors. See, generally,“Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, From Neuron ToBrain (3^(rd) Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc.(1992). Assays that detect IP₃ accumulation can be utilized to determineif a candidate compound is, e.g., an inverse agonist to a Gq- orGo-associated receptor (i.e., such a compound would decrease the levelsof IP3). Gq-dependent receptors can also been examined using an APIreporter assay in that Gq-dependent phospholipase C causes activation ofgenes containing API elements; thus, activated Gq-associated receptorswill evidence an increase in the expression of such genes, wherebyinverse agonists thereto will evidence a decrease in such expression,and agonists will evidence an increase in such expression. Commerciallyavailable assays for such detection are available.

3. GPCR Fusion Protein

The use of an endogenous, constitutively activated orphan GPCR, or anon-endogenous, constitutively activated orphan GPCR, for screening ofcandidate compounds for the direct identification of inverse agonists,agonists and partial agonists provides a unique challenge in that, bydefinition, the receptor is active even in the absence of an endogenousligand bound thereto. Thus, it is often useful that an approach beutilized that can enhance the signal obtained by the activated receptor.A preferred approach is the use of a GPCR Fusion Protein.

Generally, once it is determined that a GPCR is or has beenconstitutively activated, using the assay techniques set forth above (aswell as others), it is possible to determine the predominant G proteinthat couples with the endogenous GPCR. Coupling of the G protein to theGPCR provides a signaling pathway that can be assessed. Because it ismost preferred that screening take place by use of a mammalianexpression system, such a system will be expected to have endogenous Gprotein therein. Thus, by definition, in such a system, theconstitutively activated orphan GPCR will continuously signal. In thisregard, it is preferred that this signal be enhanced such that in thepresence of, e.g., an inverse agonist to the receptor, it is more likelythat it will be able to more readily differentiate, particularly in thecontext of screening, between the receptor when it is contacted with theinverse agonist.

The GPCR Fusion Protein is intended to enhance the efficacy of G proteincoupling with the GPCR. The GPCR Fusion Protein is preferred forscreening with a non-endogenous, constitutively activated GPCR becausesuch an approach increases the signal that is most preferably utilizedin such screening techniques, although the GPCR Fusion Protein can alsobe (and preferably is) used with an endogenous, constitutively activatedGPCR. This is important in facilitating a significant “signal to noise”ratio; such a significant ratio is import preferred for the screening ofcandidate compounds as disclosed herein.

The construction of a construct useful for expression of a GPCR FusionProtein is within the purview of those having ordinary skill in the art.Commercially available expression vectors and systems offer a variety ofapproaches that can fit the particular needs of an investigator. Thecriteria of importance for such a GPCR Fusion Protein construct is thatthe GPCR sequence and the G protein sequence both be in-frame(preferably, the sequence for the GPCR is upstream of the G proteinsequence) and that the “stop” codon of the GPCR must be deleted orreplaced such that upon expression of the GPCR, the G protein can alsobe expressed. The GPCR can be linked directly to the G protein, or therecan be spacer residues between the two (preferably, no more than about12, although this number can be readily ascertained by one of ordinaryskill in the art). We have a preference (based upon convenience) of useof a spacer in that some restriction sites that are not used will,effectively, upon expression, become a spacer. Most preferably, the Gprotein that couples to the GPCR will have been identified prior to thecreation of the GPCR Fusion Protein construct. Because there are only afew G proteins that have been identified, it is preferred that aconstruct comprising the sequence of the G protein (i.e., a universal Gprotein construct) be available for insertion of an endogenous GPCRsequence therein; this provides for efficiency in the context oflarge-scale screening of a variety of different endogenous GPCRs havingdifferent sequences.

E. Other Utility

Although a preferred use of the human orphan GPCRs disclosed herein maybe for the direct identification of candidate compounds as inverseagonists, agonists or partial agonists (preferably for use aspharmaceutical agents), these versions of human GPCRs can also beutilized in research settings. For example, in vitro and in vivo systemsincorporating GPCRs can be utilized to further elucidate and understandthe roles these receptors play in the human condition, both normal anddiseased, as well as understanding the role of constitutive activationas it applies to understanding the signaling cascade. The value in humanorphan GPCRs is that its utility as a research tool is enhanced in thatby determining the location(s) of such receptors within the body, theGPCRs can be used to understand the role of these receptors in the humanbody before the endogenous ligand therefor is identified. Other uses ofthe disclosed receptors will become apparent to those in the art basedupon, inter alia, a review of this patent document.

Although a preferred use of the non-endogenous versions of the humanRUP3 disclosed herein may be for the direct identification of candidatecompounds as inverse agonists, agonists or partial agonists (preferablyfor use as pharmaceutical agents), this version of human RUP3 can alsobe utilized in research settings. For example, in vitro and in vivosystems incorporating RUP3 can be utilized to further elucidate theroles RUP3 plays in the human condition, particularly with respect tothe human pancreas, both nonnal and diseased (and in particular,diseases involving regulation of insulin or glucagon, e.g., diabetes),as well as understanding the role of constitutive activation as itapplies to understanding the signaling cascade. A value innon-endogenous human RUP3 is that its utility as a research tool isenhanced in that, because of its unique features, non-endogenous RUP3can be used to understand the role of RUP3 in the human body before theendogenous ligand therefor is identified. Other uses of the disclosedreceptors will become apparent to those in the art based upon, interalia, a review of the patent document.

EXAMPLES

The following examples are presented for purposes of elucidation, andnot limitation, of the present invention. While specific nucleic acidand amino acid sequences are disclosed herein, those of ordinary skillin the art are credited with the ability to make minor modifications tothese sequences while achieving the same or substantially similarresults reported below. Unless otherwise indicated below, all nucleicacid sequences for the disclosed endogenous orphan human GPCRs have beensequenced and verified. For purposes of equivalent receptors, those ofordinary skill in the art will readily appreciate that conservativesubstitutions can be made to the disclosed sequences to obtain afunctionally equivalent receptor.

Example 1

Endogenous Human GPCRs

1. Identification of Human GPCRs

Several of the disclosed endogenous human GPCRs were identified basedupon a review of the GenBank database information. While searching thedatabase, the following cDNA clones were identified as evidenced below.

Open Disclosed Complete Reading Nucleic Amino Human DNA Frame Acid AcidOrphan Accession Sequence (Base SEQ ID. SEQ ID. GPCRs Number (BasePairs) Pairs) NO. NO. hARE-3 AL033379 111,389 bp 1,260 bp 1 16 hARE-4AC006087 226,925 bp 1,119 bp 3 4 hARE-5 AC006255 127,605 bp 1,104 bp 5 6hRUP3 AL035423 140,094 bp 1,005 bp 7 8 hRUP5 AC005849 169,144 bp 1,413bp 9 10 hRUP6 AC005871 218,807 bp 1,245 bp 11 12 hRUP7 AC007922 158,858bp 1,173 bp 13 14

Other disclosed endogenous human GPCRs were identified by conducting aBLAST search of EST database (dbest) using the following EST clones asquery sequences. The following EST clones identified were then used as aprobe to screen a human genomic library.

Open Nucleic Amino Disclosed Reading Acid Acid Human EST Clone/ FrameSEQ SEQ Orphan Query Accession No. (Base ID. ID. GPCRs (Sequence)Identified Pairs) NO. NO. hGPCR27 Mouse AA775870 1,125 bp 15 16 GPCR27hARE-1 TDAG 1689643   999 bp 17 18 AI090920 hARE-2 GPCR27 68530 1,122 bp19 20 AA359504 hPPR1 Bovine 238667 1,053 bp 21 22 PPR1 H67224 hG2A MouseSee Example 1,113 bp 23 24 1179426 2(a) below hCHN3 N.A. EST 36581 1,113bp 25 26 (full length) hCHN4 TDAG 1184934 1,077 bp 27 28 AA804531 hCHN6N.A. EST 2134670 1,503 bp 29 30 (full length) hCHN8 KIAA0001 EST 764451,029 bp 31 32 hCHN9 1365839 EST 1541536 1,077 bp 33 34 hCHN10 Mouse ESTHuman 1,005 bp 35 36 1365839 1365839 hRUP4 N.A. AI307658 1,296 bp 37 39N.A. = “not applicable”

2. Full Length Cloning

a. hG2A (Seq. Id. Nos. 23 & 24)

Mouse EST clone 1179426 was used to obtain a human genomic clonecontaining all but three amino acid hG2A coding sequences. The 5′end ofthis coding sequence was obtained by using 5′RACE™, and the template forPCR was Clontech's Human Spleen Marathon-ready™ cDNA. The disclosedhuman G2A was amplified by PCR using the G2A cDNA specific primers forthe first and second round PCR as shown in SEQ. ID. NO.: 39 and SEQ. ID.NO.: 40 as follows:

-   -   5′-CTGTGTACAGCAGTTCGCAGAGTG-3′(SEQ. ID. NO.: 39; 1^(st) round        PCR)    -   5′-GAGTGCCAGGCAGAGCAGGTAGAC-3′(SEQ. ID. NO.: 40; second round        PCR).

PCR was performed using Advantage™ GC Polymerase Kit (Clontech;manufacturing instructions will be followed), at 94° C. for 30 secfollowed by 5 cycles of 94° C. for 5 sec and 72° C. for 4 min; and 30cycles of 94° for 5 sec and 70° for 4 min. An approximate 1.3 Kb PCRfragment was purified from agarose gel, digested with Hind III and Xba Iand cloned into the expression vector pRC/CMV2 (Invitrogen). Thecloned-insert was sequenced using the T7 Sequenase™ kit (USB Amersham;manufacturer instructions will be followed) and the sequence wascompared with the presented sequence. Expression of the human G2A willbe detected by probing an RNA dot blot (Clontech; manufacturerinstructions will be followed) with the P³²-labeled fragment.

b. hCHN9 (Seq. Id. Nos. 33 & 34)

Sequencing of the EST clone 1541536 indicated that hCHN9 is a partialcDNA clone having only an initiation codon; ie., the termination codonwas missing. When hCHN9 was used to “blast” against the data base (nr),the 3′ sequence of hCHN9 was 100% homologous to the 5′ untranslatedregion of the leukotriene B4 receptor cDNA, which contained atermination codon in the frame with hCHN9 coding sequence. To determinewhether the 5′ untranslated region of LTB4R cDNA was the 3′ sequence ofhCHN9, PCR was performed using primers based upon the 5′ sequenceflanking the initiation codon found in hCHN9 and the 3′ sequence aroundthe termination codon found in the LTB4R 5′ untranslated region. The 5′primer sequence utilized was as follows:

-   -   5′-CCCGAATTCCTGCTFGCTCCCAGCTTGGCCC-3′ SEQ. ID. NO.: 41; sense)        and    -   5′-TGTGGATCCTGCTGTCAAAGGTCCCATTCCGG-3′ (SEQ. ID. NO.: 42;        antisense).        PCR was performed using thymus cDNA as a template and rTth        polymerase (Perkin Elmer) with the buffer system provided by the        manufacturer, 0.25 uM of each primer, and 0.2 mM of each 4        nucleotides. The cycle condition was 30 cycles of 94° C. for 1        min, 65° C. for 1 min and 72° C. for 1 min and 10 sec. A 1.1 kb        fragment consistent with the predicted size was obtained from        PCR. This PCR fragment was subcloned into pCMV (see below) and        sequenced (see, SEQ. ID. NO.: 33).

c. hRUP4 (Seq. Id. Nos. 37 & 38)

The full length hRUP4 was cloned by RT-PCR with human brain cDNA(Clontech) as templates:

-   -   5′-TCACAATGCTAGGTGTGGTC-3′ (SEQ. ID. NO.: 43; sense) and    -   5′-TGCATAGACAATGGGATTACAG-3′ (SEQ. ID. NO.: 44; antisense).        PCR was performed using TaqPlus™ Precision™ polymerase        (Stratagene; manufacturing instructions will be followed) by the        following cycles: 94° C. for 2 min; 94° C. 30 sec; 55° C. for 30        sec, 72° C. for 45 sec, and 72° C. for 10 min. Cycles 2 through        4 were repeated 30 times.

The PCR products were separated on a 1% agarose gel and a 500 bp PCRfragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen)and sequenced using the T7 DNA Sequenase™ kit (Amsham) and the SP6/T7primers (Stratagene). Sequence analysis revealed that the PCR fragmentwas indeed an alternatively spliced form of AI307658 having a continuousopen reading frame with similarity to other GPCRs. The completedsequence of this PCR fragment was as follows:

5′-TCACAATGCTAGGTGTGGTCTGGCTGGTG (SEQ. ID. NO.: 45)GCAGTCATAGTAGGATCACCATGTGGCACGTG CAACAACTTGAGATCAAATCTGACTTCCTATATGAAAAGGAACACATCTGCTGCTTAGAAGAGT GGACCAGCCCTGTGCACCAGAAGATCTACACCACCTTCATCCTTGTCATCCTCTTCCTCCTGCC TCTTATGGTGATGCTTATTCTGTACGTAAAATTGGTTATGAACTTTGGATAAAGAAAAGAGTTG GGGATGGTTCAGTGCTTCGAACTATTCATGGAAAAGAAATGTCCAAAATAGCCAGGAAGAAGAA ACGAGCTGTCATTATGATGGTGACAGTGGTGGCTCTCTTTGCTGTGTGCTGGGCACCATTCCAT GTTGTCCATATGATGATTGAATACAGTAATTTTGAAAAGGAATATGATGATGTCACAATCAAGA TGATTTTTGATATCGTGCAAATTATTGGATTTTCCAACTCCATCTGTAATCCCATTGTCTATGC A-3′

Based on the above sequence, two sense oligonucleotide primer sets:

(SEQ. ID. NO.: 46; oligo 1) 5′-CTGCTTAGAAGAGTGGACCAG-3′ (SEQ. ID. NO.:47; oligo 7) 5′-CTGTGCACGAGAAGATCTACAC-3′ and two antisenseoligonucleotide primer sets: (SEQ. ID. NO.: 48; oligo 3)5′-CAAGGATGAAGGTGGTGTAGA-3′ (SEQ. ID. NO.: 49; oligo 4)5′-GTGTAGATCTTCTGGTGCACAGG-3′were used for 3′-and 5′-race PCR with a human brain Marathon-Ready™ cDNA(Clontech, Cat# 7400-1) as template, according to manufacture'sinstructions. DNA fragments generated by the RACE PCR were cloned intothe pCRII-TOPO™ vector (Invitrogen) and sequenced using the SP6/T7primers (Stratagene) and some internal primers. The 3′ RACE productcontained a poly(A) tail and a completed open reading frame ending at aTAA stop codon. The 5′ RACE product contained an incomplete 5′ end;i.e., the ATG initiation codon was not present.

Based on the new 5′ sequence, oligo 3 and the following primer:

-   -   5′-GCAATGCAGGTCATAGTGAGC-3′ (SEQ. ID. NO.: 50; oligo 5)        were used for the second round of 5′ RACE PCR and the PCR        products were analyzed as above. A third round of 5′ RACE PCR        was carried out utilizing antisense primers:    -   5′-TGGAGCATGGTGACGGGAATGCAGAAG-3′ (SEQ. ID. NO.: 51; oligo 6)        and    -   5′-GTGATGAGCAGGTCACTGAGCGCCAAG-3′ (SEQ. ID. NO.: 52; oligo7).        The sequence of the 5′ RACE PCR products revealed the presence        of the initiation codon ATG, and further round of 5′ RACE PCR        did not generate any more 5′ sequence. The completed 5′ sequence        was confirmed by RT-PCR using sense primer        5′-GCAATGCAGGCGCTTAACATFAC-3′ (SEQ. ID. NO.: 53; oligo 8)        and oligo 4 as primers and sequence analysis of the 650 bp PCR        product generated from human brain and heart cDNA templates        (Clontech, Cat# 7404-1). The completed 3′ sequence was confirmed        by RT-PCR using oligo 2 and the following antisense primer:    -   5′-TTGGGTTACAATCTGAAGGGCA-3′ (SEQ. ID. NO.: 54; oligo 9)        and sequence analysis of the 670 bp PCR product generated from        human brain and heart cDNA templates. (Clontech, Cat# 7404-1).

d. hRUP5 (Seq. Id. Nos. 9 & 10)

The full length hRUP5 was cloned by RT-PCR using a sense primer upstreamfrom ATG, the initiation codon (SEQ. ID. NO.: 55), and an antisenseprimer containing TCA as the stop codon (SEQ. ID. NO.: 56), which hadthe following sequences:

5′-ACTCCGTGTCCAGCAGGACTCTG-3′ (SEQ. ID. NO.: 55)5′-TGCGTGTTCCTGGACCCTCACGTG-3′ (SEQ. ID. NO.: 56)and human peripheral leukocyte cDNA (Clontech) as a template. AdvantagecDNA polymerase (Clontech) was used for the amplification in a 50 ulreaction by the following cycle with step 2 through step 4 repeated 30times: 94° C. for 30 sec: 94° for 15 sec; 69° for 40 sec; 72° C. for 3min; and 72° C. from 6 min. A 1.4 kb PCR fragment was isolated andcloned with the pCRII-TOPO™ vector (Invitrogen) and completely sequencedusing the T7 DNA Sequenase™ kit (Amsham). See, SEQ. ID. NO.: 9.

e. hRUP6 (Seq. Id. Nos. 11 & 12)

The full length hRUP6 was cloned by RT-PCR using primers:

(SEQ. ID. NO.: 57) 5′-CAGGCCTTGGATTTTAATGTCAGGGATGG-3′ and (SEQ. ID.NO.: 58) 5′-GGAGAGTCAGCTCTGAAAGAATTCAGG-3′;and human thymus Marathon-Ready™ cDNA (Clontech) as a template.Advantage cDNA polymerase (Clontech, according to manufacturer'sinstructions) was used for the amplification in a 50 ul reaction by thefollowing cycle: 94° C. for 30sec; 94° C. for 5 sec; 66° C. for 40sec;72° C. for 2.5 sec and 72° C. for 7 min. Cycles 2 through 4 wererepeated 30 times. A 1.3 Kb PCR fragment was isolated and cloned intothe pCRII-TOPO™ vector (Invitrogen) and completely sequenced (see, SEQ.ID. NO.: 11) using the ABI Big Dye Terminator™ kit (P.E. Biosystem).

f. hRUP7 (Seq. Id. Nos. 13 & 14)

The full length RUP7 was cloned by RT-PCR using primers:

(SEQ. ID. NO.: 59; sense) 5′-TGATGTGATGCCAGATACTAATAGCAC-3′ and (SEQ.ID. NO.: 60; antisense) 5′-CCTGATTCATTTAGGTGAGATTGAGAC-3′and human peripheral leukocyte cDNA (Clontech) as a template. Advantage™cDNA polymerase (Clontech) was used for the amplification in a 50 ulreaction by the following cycle with step 2 to step 4 repeated 30 times:94° C. for 2 minutes; 94° C. for 15 seconds; 60° C. for 20 seconds; 72°C. for 2 minutes; 72° C. for 10 minutes. A 1.25 Kb PCR fragment wasisolated and cloned into the pCRII-TOPO™ vector (Invitrogen) andcompletely sequenced using the ABI Big Dye Terminator™ kit (P.E.Biosystem). See, SEQ. ID. NO.: 13.

g. hARE-5 (Seq. Id. Nos. 5 & 6)

The full length hARE-5 was cloned by PCR using the hARE5 specificprimers 5′-CAGCGCAGGGTGAAGCCTGAGAGC-3′ SEQ. ID. NO.: 69 (sense, 5′ ofinitiation codon ATG) and 5′-GGCACCTGCTGTGACCTGTGCAGG-3′ SEQ. ID. NO.:70 (antisense, 3′ of stop codon TGA) and human genomic DNA as template.TaqPlus Precision™ DNA polymerase (Stratagene) was used for theamplification by the following cycle with step 2 to step 4 repeated 35times: 96° C., 2 minutes; 96° C., 20 seconds; 58° C., 30 seconds; 72° C,2 minutes; and 72° C., 10 minutes

A 1.1 Kb PCR fragment of predicated size was isolated and cloned intothe pCRII-TOPO™ vector (Invitrogen) and completely sequenced (SEQ. ID.NO.: 5) using the T7 DNA Sequenase™ kit (Amsham).

h. hARE-4 (Seq. Id. Nos.: 3 & 4)

The full length hARE-4 was cloned by PCR using the hARE-4 specificprimers 5′-CTGGTGTGCTCCATGGCATCCC-3′ SEQ.ID.NO.:67 (sense, 5′ ofinitiation condon ATG) and 5′-GTAAGCCTCCCAGAACAGAGG-3′ SEQ. ID. NO.: 68(antisense, 3′ of stop codon TGA) and human genomic DNA as template. TaqDNA polymerase (Stratagene) and 5% DMSO was used for the amplificationby the following cycle with step 2 to step 3 repeated 35 times: 94° C.,3 minutes; 94° C., 30 seconds; 59° C., 2 minutes; 72° C., 10 minute

A 1.12 Kb PCR fragment of predicated size was isolated and cloned intothe pCRII-TOPO™ vector (Invitrogen) and completely sequenced (SEQ. ID.NO.: 3) using the T7 DNA Sequenase™ kit (Amsham).

i. hARE-3 (Seq. Id. Nos.: 1 & 2)

The full length hARE-3 was cloned by PCR using the hARE-3 specificprimers 5′-gatcaagcttCCATCCTACTGAAACCATGGTC-3′ SEQ.ID.NO65 (sense, lowercase nucleotides represent Hind III overhang, ATG as initiation codon)and 5′-gatcagatctCAGTT CCAATATTCACACCACCGTC-3′ SEQ. ID. NO.: 66(antisense, lower case nucleotides represent Xba I overhang, TCA as stopcodon) and human genomic DNA as template. TaqPlus Precision™ DNApolymerase (Stratagene) was used for the amplification by the followingcycle with step 2 to step 4 repeated 35 times: 94° C., 3 minutes; 94°C., 1 minute; 55° C., 1 minute; 72° C., 2 minutes; 72° C., 10 minutes.

A 1.3 Kb PCR fragment of predicated size was isolated and digested withHind III and Xba I, cloned into the pRC/CMV2 vector (Invitrogen) at theHind III and Xba I sites and completely sequenced (SEQ. ID. NO.: 1)using the T7 DNA Sequenase™ kit (Amsham).

j. hRUP3 (Seq. Id. Nos.: 7 & 8)

The full length hRUP3 was cloned by PCR using the hRUP3 specific primers5′-GTCCTGCCACTTCGAGACATGG-3′ SEQ. ID.NO.:71 (sense, ATG as intiationcodon) and 5′-GAAACTTCTCTCTGCCCTTACCGTC-3′

SEQ.ID.NO.:72 (antisense, 3′ of stop codon TAA) and human genomic DNA astemplate. TaqPlus Precision™ DNA polymerase (Stratagene) was used forthe amplification by the following cycle with step 2 to step 4 repeated35 times: 94° C., 3 minutes; 94° C., 1 minute; 58° C., 1 minute; 72° C.,2 minutes: 72° C., 10 minutes

A 1.0 Kb PCR fragment of predicated size was isolated and cloned intothe pCRII-TOPO™ vector (Invitrogen) and completely sequenced (SEQ. ID.NO.: 7)using the T7 DNA sequenase kit (Amsham).

Example 2

Receptor Expression

Although a variety of cells are available to the art for the expressionof proteins, it is most preferred that mammalian cells be utilized. Theprimary reason for this is predicated upon practicalities, i.e.,utilization of, e.g., yeast cells for the expression of a GPCR, whilepossible, introduces into the protocol a non-mammalian cell which maynot (indeed, in the case of yeast, does not) include thereceptor-coupling, genetic-mechanism and secretary pathways that haveevolved for mammalian systems—thus, results obtained in non-mammaliancells, while of potential use, are not as preferred as that obtainedfrom mammalian cells. Of the mammalian cells, COS-7, 293 and 293T cellsare particularly preferred, although the specific mammalian cellutilized can be predicated upon the particular needs of the artisan. Thegeneral procedure for expression of the disclosed GPCRs is as follows.

On day one, 1×10⁷293T cells per 150 mm plate were plated out. On daytwo, two reaction tubes will be prepared (the proportions to follow foreach tube are per plate): tube A will be prepared by mixing 20 μg DNA(e.g., pCMV vector, pCMV vector with receptor cDNA, etc.) in 1.2 mlserum free DMEM (Irvine Scientific, Irvine, Calif.); tube B will beprepared by mixing 120 μl lipofectamine (Gibco BRL) in 1.2 ml serum freeDMEM. Tubes A and B are admixed by inversions (several times), followedby incubation at room temperature for 30-45 min. The admixture can bereferred to as the “transfection mixture”. Plated 293T cells are washedwith 1×PBS, followed by addition of 10 ml serum free DMEM. 2.4 ml of thetransfection mixture will then be added to the cells, followed byincubation for 4 hrs at 37° C./5% CO₂. The transfection mixture was thenbe removed by aspiration, followed by the addition of 25 ml of DMEM/10%Fetal Bovine Serum. Cells will then be incubated at 37° C./5% CO₂. After72hr incubation, cells can then be harvested and utilized for analysis.

Example 3

Tissue Distribution of the Disclosed Human GPCRs

Several approaches can be used for determination of the tissuedistribution of the GPCRs disclosed herein.

1. Dot-Blot Analysis

Using a commercially available human-tissue dot-blot format, endogenousorphan GPCRs were probed for a determination of the areas where suchreceptors are localized. cDNA fragments from the GPCRs of Example 1(radiolabelled) were (or can be) used as the probe: radiolabeled probewas (or can be) generated using the complete receptor cDNA (excised fromthe vector) using a Prime-It II™ Random Primer Labeling Kit (Stratagene,#300385), according to manufacturer's instructions. A human RNA MasterBlot™ (Clontech, #7770-1) was hybridized with the endogenous human GPCRradiolabeled probe and washed under stringent conditions accordingmanufacturer's instructions. The blot was exposed to Kodak BioMax™Autoradiography film overnight at −80° C. Results are summarized forseveral receptors in Table B and C (see FIGS. 1A and 1B for a grididentifying the various tissues and their locations, respectively).Exemplary dot-blots are provided in FIGS. 2A and 2B for results derivedusing hCHN3 and hCHN8, respectively.

TABLE B Tissue Distribution ORPHAN GPCR (highest levels, relative toother tissues in the dot-blot hGPCR27 Fetal brain, Putamen, Pituitarygland, Caudate nucleus hARE-1 Spleen, Peripheral leukocytes, Fetalspleen hPPR1 Pituitary gland, Heart, salivary gland, Small intestine,Testis hRUP3 Pancreas hCHN3 Fetal brain, Putamen, Occipital cortex hCHN9Pancreas, Small intestine, Liver hCHN10 Kidney, Thyroid

TABLE C Tissue Distribution ORPHAN GPCR (highest levels, relative toother tissues in the dot-blot hARE-3 Cerebellum left, Cerebellum right,Testis, Accumbens hGPCR3 Corpus collusum, Caudate nucleus, Liver, Heart,Inter- Ventricular Septum hARE-2 Cerebellum left, Cerebellum right,Substantia hCHN8 Cerebellum left, Cerebellum right, Kidney, Lung

To ascertain the tissue distribution of hRUP3 mRNA, RT-PCR was performedusing hRUP3-specific primers and human multiple tissue cDNA panels (MTC,Clontech) as templates. Taq DNA polymerase (Stratagene) was utilized forthe PCR reaction, using the following reaction cycles in a 40 ulreaction: 94° C. for 2 min; 94° C. for 15 sec; 55° C. for 30 sec; 72° C.for 1 min: 72° C., for 10 min. Primers were as follows:

(SEQ. ID. NO.: 61; sense) 5′-GACAGGTACCTTGCCATCAAG-3′ (SEQ. ID. NO.: 62;antisense) 5′-CTGCACAATGCCAGTGATAAGG-3′.20 ul of the reaction was loaded onto a 1% agarose gel: results are setforth in FIG. 3.

As is supported by the data of FIG. 3, of the 16 human tissues in thecDNA panel utilized (brain, colon, heart, kidney, lung, ovary, pancreas,placenta, prostate, skeleton, small intestine, spleen, testis, thymusleukocyte, and liver) a single hRUP3 band is evident only from thepancreas. Additional comparative analysis of the protein sequence ofhRUP3 with other GPCRs suggest that hRUP3 is related to GPCRs havingsmall molecule endogenous ligand such that it is predicted that theendogenous ligand for hRUP3 is a small molecule.

b. hRUP4

RT-PCR was performed using hRUP4 oligo's 8 and 4 as primers and thehuman multiple tissue cDNA panels (MTC, Clontech) as templates. Taq DNApolymerase (Stratagene) was used for the amplification in a 40 ulreaction by the following cycles: 94° C. for 30 seconds, 94° C. for 10seconds, 55° C. for 30 seconds, 72° C. for 2 minutes, and 72° C. for 5minutes with cycles 2 through 4 repeated 30 times.

20 ul of the reaction were loaded on a 1% agarose gel to analyze theRT-PCR products, and hRUP4 mRNA was found expressed in many humantissues, with the strongest expression in heart and kidney. (see, FIG.4). To confirm the authenticity of the PCR fragments, a 300 bp fragmentderived from the 5′ end of hRUP4 was used as a probe for the SouthernBlot analysis. The probe was labeled with ³²P-dCTP using the Prime-ItII™ Random Primer Labeling Kit (Stratagene) and purified using theProbeQuant™ G-50 micro columns (Amersham). Hybridization was doneovernight at 42° C. following a 12 hr pre-hybridization. The blot wasfinally washed at 65° C. with 0.1×SSC. The Southern blot did confirm thePCR fragments as hRUP4.

c. hRUP5

RT-PCR was performed using the following hRUP5 specific primers:

(SEQ. ID. NO.: 63; sense) 5′-CTGACTTCTTGTTCCTGGCAGCAGCGG-3′ (SEQ. ID.NO.: 64; antisense) 5′-AGACCAGCCAGGGCACGCTGAAGAGTG-3′and the human multiple tissue cDNA panels (MTC, Clontech) as templates.Taq DNA polymerase (Stratagene) was used for the amplification in a 40ul reaction by the following cycles: 94° C. for 30 sec, 94° C. for 10sec, 62° C. for 1.5 min, 72° C. for 5 min, and with cycles 2 through 3repeated 30 times. 20 ul of the reaction were loaded on a 1.5% agarosegel to analyze the RT-PCR products, and hRUP5 mRNA was found expressedonly in the peripheral blood leukocytes (data not shown).

d. hRUP6

RT-PCR was applied to confirm the expression and to determine the tissuedistribution of hRUP6. Oligonucleotides used, based on an alignment ofAC005871 and GPR66 segments, had the following sequences:

(SEQ. ID. NO.: 73; sense) 5′-CCAACACCAGCATCCATGGCATCAAG-3′, (SEQ. ID.NO.: 74; antisense) 5′-GGAGAGTCAGCTCTGAAAGAATTCAGG-3′and the human multiple tissue cDNA panels (MTC, Clontech) were used astemplates. PCR was performed using TaqPlus Precision™ polymerase(Stratagene; manufacturing instructions will be followed) in a 40 ulreaction by the following cycles: 94° C. for 30 sec; 94° C. 5 sec; 66°C. for 40 sec, 72° C. for 2.5 min, and 72° C. for 7 min. Cycles 2through 4 were repeated 30 times.

20 ul of the reaction were loaded on a 1.2% agarose gel to analyze theRT-PCR products, and a specific 760 bp DNA fragment representing hRUP6was expressed predominantly in the thymus and with less expression inthe heart, kidney, lung, prostate small intestine and testis. (see, FIG.5).

It is intended that each of the patents, applications, and printedpublications mentioned in this patent document be hereby incorporated byreference in their entirety.

As those skilled in the art will appreciate, numerous changes andmodifications may be made to the preferred embodiments of the inventionwithout departing from the spirit of the invention. It is intended thatall such variations fall within the scope of the invention and theclaims that follow.

Although a variety of Vectors are available to those in the art, forpurposes of utilization for both endogenous and non-endogenous humanGPCRs, it is most preferred that the Vector utilized be pCMV. Thisvector was deposited with the American Type Culture Collection (ATCC) onOct. 13, 1998 (10801 University Blvd., Manassas, Va. 20110-2209 USA)under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganisms for the Purpose of PatentProcedure. The DNA was tested by the ATCC and determined to be. The ATCChas assigned the following deposit number to pCMV: ATCC #203351.

1. A method for identifying a compound for regulating insulinconcentration in the blood of a mammal comprising the steps of:contacting one or more candidate compounds with a host cell thatexpresses a receptor comprising the amino acid sequence of SEQ ID NO: 8;and measuring the ability of the compound or compounds to inhibit orstimulate said receptor, wherein said inhibition or stimulation of saidreceptor is indicative of a compound for regulating insulinconcentration in the blood of a mammal.
 2. The method of claim 1 whereinsaid compound for regulating insulin concentration in the blood of amammal is a therapeutic for treating diabetes.
 3. The method of claim 1wherein the compound for regulating insulin concentration in the bloodof a mammal is selected from agonist, partial agonist, and inverseagonist of the receptor.
 4. The method of claim 1 wherein said host cellcomprises an expression vector, said expression vector comprising apolynucleotide, said polynucleotide comprising a nucleotide sequenceencoding a polypeptide comprising the amino acid sequence of SEQ ID NO:8.
 5. The method of claim 1 where said host cell is produced by a methodcomprising: transfecting a cell with an expression vector, saidexpression vector comprising a polynucleotide, said polynucleotidecomprising a nucleotide sequence encoding a polypeptide comprising theamino acid sequence of SEQ ID NO: 8; wherein said host cell, underappropriate culture conditions, produces a polypeptide comprising saidamino acid sequence of SEQ ID NO:
 8. 6. A method for identifying acompound for regulating glucose concentration in the blood of a mammalcomprising the steps of: contacting one or more candidate compounds witha host cell that expresses a receptor comprising the amino acid sequenceof SEQ ID NO: 8; and measuring the ability of the compound or compoundsto inhibit or stimulate said receptor, wherein said inhibition orstimulation of said receptor is indicative of a compound for regulatingglucose concentration in the blood of a mammal.
 7. The method of claim 6wherein said host cell comprises an expression vector, said expressionvector comprising a polynucleotide, said polynucleotide comprising anucleotide sequence encoding a polypeptide comprising the amino acidsequence of SEQ ID NO:
 8. 8. The method of claim 6 where said host cellis produced by a method comprising: transfecting a cell with anexpression vector, said expression vector comprising a polynucleotide,said polynucleotide comprising a nucleotide sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 8; whereinsaid host cell, under appropriate culture conditions, produces apolypeptide comprising said amino acid sequence of SEQ ID NO:
 8. 9. Amethod for identifying a compound for regulating glucagon concentrationin the blood of a mammal comprising the steps of: contacting one or morecandidate compounds with a host cell that expresses a receptorcomprising the amino acid sequence of SEQ ID NO: 8; and measuring theability of the compound or compounds to inhibit or stimulate saidreceptor, wherein said inhibition or stimulation of said receptor isindicative of a compound for regulating glucagon concentration in theblood of a mammal.
 10. The method of claim 9 wherein said host cellcomprises an expression vector, said expression vector comprising apolynucleotide, said polynucleotide comprising a nucleotide sequenceencoding a polypeptide comprising the amino acid sequence of SEQ ID NO:8.
 11. The method of claim 9 where said host cell is produced by amethod comprising: transfecting a cell with an expression vector, saidexpression vector comprising a polynucleotide, said polynucleotidecomprising a nucleotide sequence encoding a polypeptide comprising theamino acid sequence of SEQ ID NO: 8; wherein said host cell, underappropriate culture conditions, produces a polypeptide comprising saidamino acid sequence of SEQ ID NO:
 8. 12. A method for identifying acompound for inhibiting or stimulating a receptor comprising: a) theamino acid sequence of SEQ ID NO: 8; b) a mutant of SEQ ID NO: 8,wherein lysine is substituted for leucine at amino acid residue 224; c)an amino acid sequence encoded by a nucleotide sequence that hybridizesto the complete complement of SEQ ID NO:7 at 42° C., followed by washingin 0.1×SSC at 65° C.; d) an amino sequence encoded by the nucleotidesequence of SEQ ID NO: 7; e) a G protein-coupled receptor having atleast 95 % identity to the amino acid sequence of SEQ ID NO: 8, whereinsaid G protein-coupled receptor is capable of modulating insulin orglucagon levels; or f) a G protein-coupled receptor encoded by anucleotide sequence having at least 95 % identity to the nucleotidesequence of SEQ ID NO:7, wherein said G protein-coupled receptor iscapable of modulating insulin or glucagon levels, comprising the stepsof: i) contacting one or more candidate compounds with a host cell ormembrane thereof, wherein said host cell or membrane expresses areceptor comprising: a) the amino acid sequence of SEQ ID NO: 8; b) amutant of SEQ ID NO: 8, wherein lysine is substituted for leucine atamino acid residue 224; c) an amino acid sequence encoded by anucleotide sequence that hybridizes to the complete complement of SEQ IDNO:7 at 42° C., followed by washing in 0.1×SSC at 65° C.; d) an aminosequence encoded by the nucleotide sequence of SEQ ID NO: 7; e) a Gprotein-coupled receptor having at least 95 % identity to the amino acidsequence of SEQ ID NO: 8, wherein said G protein-coupled receptor iscapable of modulating insulin or glucagon levels; or f) a Gprotein-coupled receptor encoded by a nucleotide sequence having atleast 95 % identity to the nucleotide sequence of SEQ ID NO:7, whereinsaid G protein-coupled receptor is capable of modulating insulin orglucagon levels; and ii) measuring the ability of the compound orcompounds to inhibit or stimulate said receptor.
 13. The method of claim12, wherein the compound is selected from agonist, partial agonist, andinverse agonist of the receptor.
 14. The method of claim 13, wherein thecompound is an agonist of the receptor.
 15. The method of claim 13,wherein the compound is a partial agonist of the receptor.
 16. Themethod of claim 13, wherein the compound is an inverse agonist of thereceptor.
 17. The method of claim 12, wherein said host cell comprisesan expression vector, said expression vector comprising apolynucleotide, said polynucleotide comprising: a) a nucleotide sequenceencoding a polypeptide comprising the amino acid sequence of SEQ ID NO:8; b) a nucleotide sequence encoding a polypeptide comprising a mutantof SEQ ID NO: 8, wherein lysine is substituted for leucine at amino acidresidue 224; c) a nucleotide sequence that hybridizes to the completecomplement of SEQ ID NO:7 at 42° C., followed by washing in 0.1×SSC at65° C.; d) the nucleotide sequence of SEQ ID NO: 7; e) a nucleotidesequence encoding a G protein-coupled receptor having at least 95 %identity to the amino acid sequence of SEQ ID NO: 8, wherein said Gprotein-coupled receptor is capable of modulating insulin or glucagonlevels; or f) a nucleotide sequence having at least 95 % identity to thenucleotide sequence of SEQ ID NO: 7, wherein said nucleotide sequenceencodes a G protein-coupled receptor capable of modulating insulin orglucagon levels.
 18. The method of claim 12, wherein said host cell isproduced by a method comprising: transfecting a cell with an expressionvector, said expression vector comprising a polynucleotide, saidpolynucleotide comprising: a) a nucleotide sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 8; b) anucleotide sequence encoding a polypeptide comprising a mutant of SEQ IDNO: 8, wherein lysine is substituted for leucine at amino acid residue224; c) a nucleotide sequence that hybridizes to the complete complementof SEQ ID NO:7 at 42° C., followed by washing in 0.1×SSC at 65° C.; d)the nucleotide sequence of SEQ ID NO: 7; e) a nucleotide sequenceencoding a G protein-coupled receptor having at least 95 % identity tothe amino acid sequence of SEQ ID NO: 8, wherein said G protein-coupledreceptor is capable of modulating insulin or glucagon levels; or f) anucleotide sequence having at least 95 % identity to the nucleotidesequence of SEQ ID NO: 7, wherein said nucleotide sequence encodes a Gprotein-coupled receptor capable of modulating insulin or glucagonlevels, wherein said host cell, under appropriate culture conditions,produces a polypeptide comprising: a) the amino acid sequence of SEQ IDNO: 8; b) a mutant of SEQ ID NO: 8, wherein lysine is substituted forleucine at amino acid residue 224; c) an amino acid sequence encoded bya nucleotide sequence that hybridizes to the complete complement of SEQID NO:7 at 42° C., followed by washing in 0.1×SSC at 65° C.; d) an aminosequence encoded by the nucleotide sequence of SEQ ID NO: 7; e) a Gprotein-coupled receptor having at least 95 % identity to the amino acidsequence of SEQ ID NO: 8, wherein said G protein-coupled receptor iscapable of modulating insulin or glucagon levels; or f) a Gprotein-coupled receptor encoded by a nucleotide sequence having atleast 95 % identity to the nucleotide sequence of SEQ ID NO:7, whereinsaid G protein-coupled receptor is capable of modulating insulin orglucagon levels.
 19. The method of claim 12, wherein the receptorcomprises the amino acid sequence of SEQ ID NO:
 8. 20. The method ofclaim 12, wherein the receptor is a mutant of SEQ ID NO: 8, whereinlysine is substituted for leucine at amino acid residue
 224. 21. Themethod of claim 12, wherein the ability of the compound or compounds toinhibit or stimulate said receptor is measured by measuring the activityof a second messenger.
 22. The method of claim 21, wherein the secondmessenger is selected from the group consisting of adenyl cyclase andphospholipase C.
 23. The method of claim 12, wherein the ability of thecompound or compounds to inhibit or stimulate said receptor is measuredby measuring the level of a second messenger.
 24. The method of claim23, wherein the second messenger is selected from the group consistingof cAMP, diacyl glycerol, and inositol 1,4,5-triphosphate.
 25. Themethod of claim 12, wherein the ability of the compound or compounds toinhibit or stimulate said receptor is measured by measuring the bindingof GTPγS to a membrane comprising said G protein-coupled receptor. 26.The method of claim 12, wherein the host cell is a mammalian host cell.27. The method of claim 12, wherein the host cell is a yeast host cell.28. The method of claim 12, wherein the host cell comprises a reportersystem comprising multiple cAMP responsive elements operably linked to areporter gene.
 29. The method of claim 12, wherein said receptor is aconstitutively activated receptor.
 30. The method according to claim 12,wherein said method comprises identifying a compound for inhibiting orstimulating a receptor comprising: a) a G protein-coupled receptorhaving at least 98 % identity to the amino acid sequence of SEQ ID NO:8, wherein said G protein-coupled receptor is capable of modulatinginsulin or glucagon levels; or b) a G protein-coupled receptor encodedby a nucleotide sequence having at least 98 % identity to the nucleotidesequence of SEQ ID NO:7, wherein said G protein-coupled receptor iscapable of modulating insulin or glucagon levels, comprising the stepsof: contacting one or more candidate compounds with a host cell ormembrane thereof, wherein said host cell or membrane expresses areceptor comprising: a) a G protein-coupled receptor having at least 98% identity to the amino acid sequence of SEQ ID NO: 8, wherein said Gprotein-coupled receptor is capable of modulating insulin or glucagonlevels; or b) a G protein-coupled receptor encoded by a nucleotidesequence having at least 98 % identity to the nucleotide sequence of SEQID NO:7, wherein said G protein-coupled receptor is capable ofmodulating insulin or glucagon levels, and measuring the ability of thecompound or compounds to inhibit or stimulate said receptor.
 31. Themethod of claim 17, wherein said host cell comprises an expressionvector, said expression vector comprising a polynucleotide, saidpolynucleotide comprising: a) a nucleotide sequence encoding a Gprotein-coupled receptor having at least 98 % identity to the amino acidsequence of SEQ ID NO: 8, wherein said G protein-coupled receptor iscapable of modulating insulin or glucagon levels; or b) a nucleotidesequence having at least 98 % identity to the nucleotide sequence of SEQID NO: 7, wherein said nucleotide sequence encodes a G protein-coupledreceptor capable of modulating insulin or glucagon levels.
 32. Themethod of claim 18, wherein said host cell is produced by a methodcomprising: transfecting a cell with an expression vector, saidexpression vector comprising a polynucleotide, said polynucleotidecomprising: a) a nucleotide sequence encoding a G protein-coupledreceptor having at least 98 % identity to the amino acid sequence of SEQID NO: 8, wherein said G protein-coupled receptor is capable ofmodulating insulin or glucagon levels; or b) a nucleotide sequencehaving at least 98 % identity to the nucleotide sequence of SEQ ID NO:7, wherein said nucleotide sequence encodes a G protein-coupled receptorcapable of modulating insulin or glucagon levels, wherein said hostcell, under appropriate culture conditions, produces a polypeptidecomprising: a) a G protein-coupled receptor having at least 98 %identity to the amino acid sequence of SEQ ID NO: 8, wherein said Gprotein-coupled receptor is capable of modulating insulin or glucagonlevels; or b) a G protein-coupled receptor encoded by a nucleotidesequence having at least 98 % identity to the nucleotide sequence of SEQID NO:7, wherein said G protein-coupled receptor is capable ofmodulating insulin or glucagon levels.